Vascular stiffening in aging females with a hypertension‐induced HIF2A gain‐of‐function mutation

Abstract Pulmonary arterial hypertension (PAH) is more prevalent in females than males; the causes of this sex difference have not been adequately explored. Gain‐of‐function (GOF) mutations in hypoxia‐inducible factor 2α (HIF2A) lead to PAH and thrombotic consequences in patients and mice. Additionally, multiple emerging studies suggest that elevated systemic arterial stiffening (SAS) occurs in PAH; this could have critical prognostic value. Here, we utilized a HIF2A GOF mouse model to determine how SAS can be used as a prognosticator in sex‐divergent PAH. We analyzed survival, vascular mechanics, and vascular phenotypes in young adult (8–16 weeks) and middle age (9–12 months) Hif2a GOF mice. We find that Hif2a heterozygous (HT) female mice, but not Hif2a HT male mice, exhibit poor survival, SAS upon aging, and decreased ability to withstand repeated physiological strain. Hif2a HT female mice also display thickening of the adventitial intima and increased collagen I and collagen III in all layers of the thoracic aorta. Our findings demonstrate differing PAH progression in female and male Hif2a GOF mice. Specifically, alterations in extracellular matrix (ECM) content led to vascular stiffening in aged females, resulting in poor survival. Moreover, we show that SAS emerges early in mice with PAH by coupling studies of vascular mechanics and analyzing vascular structure and composition. Importantly, we present a model for assessing sex differences in hereditary PAH progression and sex‐specific prognosis, proposing that aortic stiffening can be used to prognosticate future poor outcomes in PAH.

to changes in the extracellular matrix (ECM) quantity and the composition of each layer.
The estimated incidence of PAH is 1.1-7.6 per million adults per year, with a prevalence of 6.6-26.0 per million adults per year. [10][11][12] Neither idiopathic nor heritable PAH is sex-blind; both predominately affect females. [13][14][15] Interestingly, the balance of PAH incidence in the two sexes changes with age. The COMPERA European registry showed that the overall 1.8:1 female-to-male ratio fell to 1.2:1 when limited to patients older than 65. 16 Moreover, PAH is most commonly diagnosed in women between the ages of 30-60, while males are often diagnosed with PAH at older ages. 17 This asymmetric relationship concerning the time of initial diagnosis and incidence is inadequately understood. To address the gap in our understanding of the differences in prevalence and incidence of PAH in male and female patients, we aimed to examine PAH progression in the context of sex and age.
Accumulating evidence shows that PAH may also cause systematic arterial stiffness (SAS). 18,19 Multiple prior clinical studies have correlated increases in SAS with PAH patient disease progression. [20][21][22] Specifically, increased vascular stiffness is positively associated with age and disease progression and negatively correlated with survival. [20][21][22] Nonetheless, the prognostic value of vascular stiffness remains incompletely understood but points to the possibility of PAH as a cause of accelerated arterial aging or to the potential for vascular interdependency. 23,24 Thus, in the present study, we sought to determine if PAH pathogenesis accelerates the deterioration of systemic vascular mechanics with age and whether this links to mouse sex.
Hypoxia-inducible factor (HIF) signaling plays a fundamental role in PAH pathogenesis. 25 HIF activity is regulated in an oxygendependent manner; degraded in normal oxygen conditions, HIFs stabilize and accumulate in hypoxia. Multiple mutations in the HIF2A gene associated with erythrocytosis have been identified, with some patients developing pulmonary hypertension. [26][27][28][29][30] Specifically, missense mutations in H1F2A G537W impair HIF2a hydroxylation and have been shown to cause familial PAH. 30 Mice with missense Hif2a G536W mutations display mutation-dependent erythrocytosis and pulmonary hypertension. 31,32 Moreover, smooth muscle cells from mice and patients with missense H1F2A mutations exhibit increased stiffness and abnormal f-actin fiber orientation. 32 In the present study, we sought to determine whether SAS occurs in familial hypertensive aging female mice compared with male counterparts, thereby providing SAS as a prognosticator of PAH. We study the differences in the temporal evolution of HIF2A-driven PAHassociated ECM changes. Specifically, we examine whether this can be attributed to sex, a variable infrequently assessed in the PAH field.
We leverage the HIF2A GOF mouse model, previously established consistent with hereditary Hif2a G537R, and we couple survival, circumferential tensile testing (CTT), gene expression, and immunohistochemical (IHC) analyses to probe the contributions of collagens I, III, and elastin to physiological mechanical properties of the hypertensive thoracic aorta. 32 2 | RESULTS

| Female Hif2a HT and HO mice display characteristics of PAH and erythrocytosis
Previous studies have shown that mutations in G537 of the HIF2A gene cause familial hypertension. 26,31,32 Thus, we focused on a Hif2a G536W knock-in mutation in a C57BL/6 background mouse. 30 While prior studies have extensively characterized and assessed this mouse model as having pulmonary hypertension (RV pressure > 60 mm Hg in HO) and erythrocytosis, they have not confirmed the disease presentation of the female mice. 31,32 We mated Hif2a G536W/+ (heterozygote; HT) mice and obtained both Hif2a HT and Hif2a G536W/G536W (homozygote; HO) mice. Hif2a +/+ (wild-type; WT) littermates served as controls. However, all HIF2A gene mutations in humans are heterozygous 31 ; the homozygous mutation is lethal in humans.
First, we measured the mice's red blood cell (RBC) counts. The RBC count for aged female mice increased in a mutation-dose-dependent ( Figure S1A). We then measured Fulton Index and normalized heart weight for combined middle-aged and early aging female WT, Hif2a HT, and Hif2a HO mice. We observed increased normalized heart weight in aged female Hif2a WT mice (Figure S1B-C). We next measured hematocrit and hemoglobin and marked increases in aged female Hif2a HT and Hif2a HO mice ( Figure S1D,E). We found no statistically significant differences in the groups when measuring the white blood cell, platelet, and immune cell counts ( Figure S1F,G). We performed the same analysis on aged male WT, Hif2a HT, and Hif2a HO mice and found similar trends except for increased statistical significance in the lymphocyte counts ( Figure S2).
2.2 | Aging female Hif2a heterozygote and homozygote mice exhibit higher mortality than their male genetic counterparts After confirming PAH in the mouse model, we performed aging studies to elucidate changes in the distribution of genotypes and their likelihood of survival to 12 months. Consistent with prior studies, Hif2a HO mice were born at a lower frequency than expected according to the Mendelian genetics (data not shown), suggesting the Hif2a HO mice have a survival disadvantage from birth. 31 Female mice exhibited mutation dose-dependent differences in their mortality trends; a phenomenon that was not seen for the male mice ( Figure 1). When directly compared, Hif2a HT female mice trended toward higher mortality than Hif2a HT male mice compared to their WT counterparts ( Figure S3). In addition, our data suggest that female Hif2a HT and HO, mice have a disadvantage in survival with aging. Thus, we focused our study on female mice. Given the precipitous increase in mortality in the PAH group at $13 months of age when compared with WT littermates, we defined the age groups of interest as being: young adult for 8-16 weeks, early middle age for 4-8 months, middle age for 9-12 months, and early aging for 13 months+. Our studies focused on a direct comparison of the young adult and middle age groups, as survival in the early aging group provided limited subjects for comparison.

| Middle age female Hif2a HT mice have stiffer thoracic aorta than their young adult counterparts
Emerging studies link SAS to PAH. Therefore, we next sought to examine whether female WT and Hif2a HT mice had any alterations in their response to functional tensile stress with aging; thus, we examined the response of the thoracic aorta using CTT. We focused on the young adult (8-16 weeks) and middle-aged (9-12 months) age groups, which correlate approximately to young adolescence/young adulthood (13-20 years) and middle ages (30-40 years) in humans, respectively. 33 To assess the contribution of the ECM to the mechanical properties, we decellularized the thoracic aortas and measured the mechanical properties of cellular and acellular segments of the same thoracic aorta. CTT data were represented by the equation S = α exp (βλ), where α and β are constants determined by least squares curve fitting. We found that middle-aged female Hif2a HT mice exhibit vascular stiffening, as evidenced by the left shift in the stress versus strain curve (Figures 2ai,bi and S4A,B). Aging female WT mice did not exhibit a similar trend, suggesting that the vascular stiffening phenotype is specific to the PAH mice. We examined the maximum tensile stress for female WT and Hif2a HT mice and found no statistically significant differences (Figure 2aii,bii). Finally, we examined strain at failure (Figure 2aiii,biii) and found a dramatic decrease between female Hif2a HT acellular young adult and middle age segments' strain at failure, suggesting that thoracic aortas of aged female Hif2a HT mice experience more significant changes in their ECM and that these changes dramatically impact their ability to handle physiological strain To adequately assess sex differences in how PAH evolves with HIF2a GOF, we performed the preceding experiments with male mice.
Here too, we decellularized the thoracic aortas and measured cellular and acellular segments of proximal aortic segments. We found that middle age male WT and Hif2a HT mice are less capable of withstanding repeated physiological stresses than their young adult counterparts, evident by the right shift in the stress versus strain curve ( Figure 3ci,di). We also examined the maximum tensile stress of the thoracic aorta from male WT and Hif2a HT mice and found no statistically significant differences (Figure 3cii,dii). Finally, we examined strain at failure. Interestingly, we found statistically significant differences between young adult and middle age cellular segments of male WT, a difference we did not see in the female counterparts ( Figure 3ciii).

| Middle age female Hif2a
HT mice exhibit thoracic aorta adventitial layer thickening compared to WT counterparts Next, we examined the differences in the ECM by immunohistochemistry (IHC) analysis of thoracic aortic rings. As the middle age Hif2a HT female mice exhibited vascular stiffening and statistically meaningful differences in their strain at failure compared to young adult Hif2a HT female mice, we focused on these two age groups. Littermate female WT counterparts served as controls. We completed Masson's trichrome stain (MAS) and Verhoeff-Van Giesson (VVG) staining and quantified the thickness of the adventitial layer, medial layer, and the adventitial/medial layer thickness ratio for young adult and middle age female WT and Hif2a HT mice (Figure 3a lamellae, lamellar thickness, and interlamellar distance ( Figure S5).
While young adult and middle-aged mice had different lamellar thickness, no statistically significant differences were found between the WT and Hif2a HT mice. Thus, we further focused on the major ECM components of the adventitial layer, the collagens, and their differences across our groups of interest. 2.5 | Collagen I and collagen III accumulate in the aortas of middle age female HT mice compared to young adult female counterparts First, we examined differences in the ECM components by performing qRT-PCR on mouse abdominal aortas (Figure 4a). We did not find any statistically significant differences in levels of ELN, COL1A1, and COL3A1 transcripts. We next evaluated collagens I and III accumulation by performing IHC for collagen I and collagen III on young adult and middle age WT and HT female mouse thoracic aortas. We found that Hif2a HT female mice have more collagens I and III than WT female mice (Figure 4b; Figure S6).

| DISCUSSION
PAH is more prevalent in female patients than in male patients. While the causes of this sex difference have not been adequately explored, clinical database analyses have suggested several potential avenues. [34][35][36] Researchers have found that female PAH positively correlates with patient use of prescription weight-loss drugs, recreational drugs, and oral contraceptive pills. 17 Researchers have also suggested that endogenous sex hormones, specifically 17β oestradiol and its metabolites, could cause increased female PAH incidence. 37 Finally, some studies hypothesize that the higher incidence of autoimmune diseases (including systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, thyroiditis) in females, when further associated with PAH, has the potential to increase inflammation and to drive disease progression. 38   We then examined the adventitial and medial layers of the thoracic aorta. Middle age female Hif2a HT mice exhibited thickening in their adventitial layer that is not seen in young adult female mice of either genotype. Surprisingly, we did not see any RNA transcript level differences between the female, young adult, and middle age WT and Hif2a HT mice. When we further examined middle age and young adult female Hif2a HT and WT mice for expression of collagens I and III, we found that middle age female Hif2a HT mice, compared to WT counterparts, have significantly greater collagen I and III deposits throughout their vessel architecture. This trend was also present in the younger female HT mice but to a lesser effect. Moreover, both middle age and young adult Hif2a HT mice exhibited a high degree of heterogeneity in their collagen I content, which may cause the large variability observed in vessel mechanics. Overall, collagens I and III depositions were both age and genotype dependent. Consequently, we conclude that elevated ECM deposition is the mechanism responsible for Hif2a HT female mouse aortic stiffening. Taken together, these findings suggest that accelerated aging of the systemic circula- We acknowledge that our study has limitations. Due to the structure and the already long timescale of the study, our Kaplan-Meier curves do not exceed 12 months. We do not address the cause of death in female PAH mice. We did not establish the causality between the Hif2a mutation and collagen deposition in the systemic vasculature. We also did not delve into sex-specific differences in PAH cells.    Table 1 for details of the major resources used in these studies. Polymerase chain reactions (PCR) on young adult tail DNA were used to identify genotype, using primers for G536W. Mice were aged until they fit one of the predetermined age ranges, being a young adult (8-16 weeks), early middle age (16 weeks-9 months), middle age (9-12 months), and early aging (13 months+). Animals were housed on a 12-h light/dark cycle and were given access to food and water ad libitum. Animals were massed, sacrificed, and tissues were immediately collected for further experiments.

| Tensile testing
Circumferential tensile testing was performed following protocols described previously 43,44 with minor modifications. 45,46 The thoracic aortas were harvested, cleaned, and cut into 1.25-1.5 mm length segments. The length of each segment was imaged using a microscope set to a magnification of Â4. The length was measured using Image J software (NIH). The rings were then mounted onto pins on an electromechanical puller (DMT560). After appropriate system calibration and pin alignment, the pins were separated using an electromotor at a 50 μm/s to apply radial force onto the specimen until breakage. Displacement and force were continuously recorded. The thickness of the intimal and medial layers (t) and stress-free lumen diameter (D i ) of representative samples were measured from concentric rings cut adjacent to the pulled segments, imaged at Â4 magnification, and quantified using Image J software. Engineering Stress (S) was calculated by normalizing force (F) to the initial stress-free area of the specimens (S=F/(2t*L)). Engineering strain (λ) was calculated as the displacement ratio to the initial stress-free diameter. The raw data was processed in Excel (Microsoft) and MATLAB (MathWorks), where an exponential fit, using least-squares fitting, was applied to the tensile testing data.
All vessel segments that did not comply with the exponential fit with a minimum coefficient of determination, R 2 of 0.95 were excluded from the exponential fit curves and from the raw data set. All calculations were done using Excel (Microsoft) and MATLAB (MathWorks).
The medial and adventitial layer thickness was measured on MASstained slides by measuring the length of each layer perpendicular to approximate vessel curvature. Ten measurements were taken per 40Â image, and a minimum of 10 40Â images were averaged per animal (n = 10+). Elastin lamellar thickness, interlamellar distance, and the number of lamellae were measured on VVG-stained slides by measuring the length of each object perpendicular to approximate vessel curvature. Ten measurements were taken per 40Â images, and a minimum of 10 40Â images were averaged per animal (n = 10+), except for the number of lamellar layers, which was measured in one location per 40Â image.
Collagen I and collagen III staining analysis was performed using a custom MATLAB code based on the MATLAB internal "Color Thresholder" app and normalized to the total area of the vessel in each 40Â image. Cohorts were protein and age matched to select ideal +positive thresholder values.

| Statistics and reproducibility
For all experiments, "n" denotes technical replicates while "N" represents biological replicates. The number of technical replicates and biological replicates varies depending on the experiment and is noted specifically in each figure legend. Two-tailed t-tests or ANOVA were performed to determine significance. All graphs were drawn using GraphPad Prism 9. Significance levels were set at *p < 0.05, **p < 0.01, and ***p < 0.001.

CONFLICT OF INTEREST
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.